Cancer is a leading cause of death in human and the number of affected individuals increases for each year. Although the different methods of treatment for cancer, e.g. chemotherapy, endocrine therapy, radiotherapy and surgery, have improved tremendously the last decades, they are far from perfect, in particular for patients with late stages of cancer. Thus, much research has been spent on early detection of tumors in patients.
One used method for tumor and tumor stage detection is determination of cell proliferation in patients. The proportion of DNA-synthesizing cells (S-phase cells) in tumors has been used as a measure of their proliferation rate. The DNA-synthesizing cells have earlier been determined by means of radioactive-labeled thymidine (autoradiography). Incorporation of halogen-analogs of thymidine (BrdU) and antibodies against these have been used together with quantitative flow-cytometric DNA measurements. The disadvantage with the use of isotopic labeled thymidine and BrdU is that only living cells can be measured. Therefore, these methods have only been used on selected patients and in a limited number of studies [Wilson, Acta Oncol, 30; 903, 1991]. Furthermore, the flow-cytometry technique is unable to distinguish between proliferating and non-proliferating cells. Determination of S-phase cells is even more complicated in tumors, since the cancer tumor cells do not deviate in their DNA-content from benign cells [Tribukait, World J. Urol, 5: 108, 1987].
Instead of measuring DNA-synthesis itself, markers related to proliferating cells has been used, for example, Ki-67 and PCNA (Proliferating Cell Nuclear Antigen). Ki-67 is expressed in all cell cycle stages except for G0 [Scholzen, J. Cellular Physiol, 182: 311, 2000]. Antibodies against Ki-67 can be used on fresh tissues as well as on formaline feed and paraffin embedded tissues. Depending on the cell proliferation rate, PCNA is expressed in all cell cycle stages. PCNA is not as sensitive for various fixation techniques as Ki-67 [Hall, Cell Tissue Kinet, 25:502, 1990]. Antibodies against other types of proteins and enzymes involved in the DNA-synthesis (DNA polymerase, ribonucleotide reductase) have been tested, but with limited successful results [Wilson, Acta Oncol., 30: 903, 1991].
Thymidine kinase (TK), an enzyme of the pyrimidine salvage pathway, catalyses the phosphorylation of thymidine to thymidine monophosphate. TK in human cells appears in two forms, a cytoplasmic (TK1) and a mitochondrial (TK2) protein, encoded by different genes. Human TK1 and TK2 are located on the chromosome 17q23.2-q25.3 and 16q22-q23.1, respectively. TK1 transcripts encode a 25.5 kDa protein with, highly conserved regions typical for nucleoside kinases. However, the crystal structures of this enzyme family have no yet been determined. The expression of TK1 is cell cycle regulated and the TK1 regulation is complex with mRNA levels peaking in proliferating cells. Splicing and translation of TK1 mRNA also varies in cells at different growth states, TK1 levels are mainly regulated by post-translational mechanisms, in particular by differential degradation due to highly active protease expression in mitotic cells. The C-terminal region of TK1 contains a specific sequence. KEN, which recently has been shown to be the signal for mitotic degradation of TK1 by the Anaphase-promoting complex/cyclosome-Cdh1-mediated pathway [Ke, Mol. Cell. Biol., 24:514, 2004], TK2 is not cell cycle regulated and is the only TK enzyme found in resting cells [Wintersberger, Biochem. Soc. Trans., 25:303, 1997; Sherley, J. Biol. Chem., 263:8350, 1988; He, Cell. Prolif., 24:3, 1991; Kauffman, Mol. Cell. Biol., 11:2538, 1991; Hengstschläger, J. Biol. Chem., 269:13836, 1994; Munch-Petersen, J. Biol Chem., 266:9032, 1991].
The majority of the above-mentioned markers have been, used to identify proliferation cells in tissues. However, thymidine kinase activity has been determined in cytosol fractions of tissues as a proliferation marker in human breast cancer. In a study of 1,692 breast, cancer patient [Broet, J. Clin. Oncol., 19:2778, 2000], high TK1 activity in the cytosol correlated to a shorter survival as well as a poor outcome of endocrine treatment (tamoxifen) [Foekens, Cancer Res., 61:1421, 2001]. Furthermore, thymidine kinase has also been used as a marker of cell proliferation in serum by measuring its enzyme activity.
Because of the tight, coupling between the serum TK (STK) enzyme activity and high proliferation, it is considered a sensitive and useful marker for cell proliferation and hence for malignancy detection [He, Internal J. Biol Marker, 15:139, 2000; Zou, Internal. J. Biol Marker, 17:135, 2002; He, Biochimica Biophysica Acta, 1289:25, 1996; He, Europ. J. Cell Biol., 70:117, 1996; Wu, Anticancer Res., 6:4867, 2000; Mao, Cancer Inves. 20:922, 2002; Wang, Analysis Cell. Pathology, 23:11, 2001; Kuroiwa, J. Immuno. Methods, 253; 1, 2001], Thus, STK enzyme activity has been, used as a turner marker in patients with different blood tumors. However, STK activity has been found to be not a good marker in patients with solid tumors.
More than 95% of STK enzyme activity corresponds to TK1 while less than 5% corresponds to TK2. The composition and the properties of STK are not yet well understood. Results indicate that STK is a polymeric form of TK1, probably as complex also with other serum proteins and it has a total molecular weight of approximately 700 kDa [Karlström, Mol. Cell. Biochem, 92:23, 1990].
Using the thymidine analogue 5-iodo-2′-deoxyuridine as substrate, STK activity was established as a serological proliferation marker in 1984[Gronowich, Br. J. Cancer, 47:487, 1983] and is now available as a commercial RIA-kit-[125I]-radio-assay (Sangtec Medical AB, Stockholm, Sweden, recently purchased by DiaSorin Inc.). The STK activity assay has been useful for estimation of tumor spread and prognosis in patients with the acute leukemia and chronic leukemia (CLL), Hodgkin and non-Hodgkin's lymphoma, but not in the case of solid tumors [Gronowich, Int. J. Cancer, 15:5, 1984]. The STK enzyme activity in CLL patient provides useful prognostic information regarding both responses to therapy and length of survival. Although the method is relatively effective, especially for leukemia and lymphoma diseases, the 5-iodo-2′-deoxyuridine is not a specific substrate for STK (TK1) activity. Furthermore, the [125I]-radio-iodo-deoxyuridine has a short half-life (four weeks), the STK engine activity is highly sensitive to temperature and pH changes, and the radioassay requires specialized equipment (γ-scintillation counter), isotope laboratory and highly skilled, personnel. The disadvantages of such a radioassay technique have probably limited the clinical use of this assay.
Therefore, & new STK activity kit based on antibodies against the product of the STK reaction has recently been developed. Anti-TK1 antibodies have also been generated during the last decades, mostly polyclonal antibodies for basic research and not for commercial use. Recently, mouse mono- and polyclonal anti-TK1 antibodies have been developed for potential clinical purpose, i.e. for breast cancer [Zhang, Cancer Detection Prev., 25:8, 2001] and lung cancer [Voeller, Anti-Cancer Drugs, 12:555, 2001]. One anti-TK1 monoclonal antibody is now commercial available for basic research, but not for clinical use (QED Bioscience Inc, San Diego, USA, 2003).
In the U.S. Pat. No. 5,698,409 O'Neil discloses monoclonal antibodies (mAb) generated against the enigmatically active 100 kDa tetrameric form of TK1 from Raji cells. The mAb are used for determining only this enzymatically active form of TK1 in biological samples from patients. Furthermore, O'Neil has to determine the TK activity using radiolabeled thymidine, in addition to using the mAb. The determined TK1 activities are then used for diagnosing and monitoring various forms of cancer. The properties of their anti-TK1 monoclonal antibodies raise questions about the specificity of these antibodies, i.e. they react with a protein of higher molecular weight than expected in SDS gelelectrophoresis Western blot (45 kDa versus 25 kDa in extracts of human HeLa cells). Furthermore, the antibodies detect a protein in the serum occurring in another (higher) concentration range than found by other workers in the field, i.e. they need to dilute serum 16,000 times while others use undiluted serum. These facts suggest that their antibodies react with other proteins in addition to enzymatically active TK1 and/or TK2. In addition, the method of O'Neil is impaired with the similar problems as with usage of the radiolabeled thymidine analogue discussed above.
Kuroiwa, et al, [Kuriowa, J. Immuno. Methods, 253:1, 2001] have developed and tested 26 anti-TK1 monoclonal antibodies. The properties of these antibodies are more as expected, i.e. they react with a protein with a molecular weight of 25 kDa in extracts of human HeLa in Western blot with no reported cross-reactivity to other proteins. When they used these antibodies in serum from patients with solid tumors using ELISA, they found no significant differences of serum TK1 levels as compared to serum TK1 levels from healthy individuals.